Fluorescence is a useful analytical technique for studying nucleic acid/nucleic acid and protein/nucleic acid interactions. In particular, fluorescent nucleosides and their analogs can be used to probe the physical and chemical environment of macromolecules to which they are covalently bonded or electrostatically attached. Various physical and chemical environmental changes can be assessed using fluorescence such as, changes in pH, interaction with other molecules, or alteration in tertiary structure. These changes can manifest differences in fluorescence intensity, the lifetime of fluorescence emission or fluorescence depolarization. In addition, there can be a shift in the emission, excitation, or absorption spectra of the fluorescent nucleoside depending on its environment. By measuring changes in fluorescence, useful information relating to macromolecule interactions, nucleic acid hybridization and enzymatic reactions can be obtained.
Fluorescent oligonucleotides find additional uses in molecular biology as probes for screening genomic and complementary DNA libraries, as primers for DNA synthesis, sequencing, and amplification reactions. Oligonucleotide probes have also proven useful for assaying in vitro gene expression using techniques of in situ hybridization. Recent improvements in DNA sequencing methods, fluorescent labels, and detection systems have dramatically increased the use of fluorescently labeled oligonucleotides in all of the foregoing applications. Typically, oligonucleotides are labeled with a fluorescent marker, either directly through a covalent linkage (e.g., a carbon linker), intercalation, or indirectly whereby the oligonucleotide is bound to a molecule such as biotin or dioxigenin, which is subsequently coupled to a fluorescently labeled binding moiety (e.g., streptavidin or a labeled monoclonal antibody).
These fluorescent labeling systems, however, suffer the disadvantage that the fluorescent complexes and their binding moieties are relatively large. The presence of large fluorescent labels and associated linkers can alter the mobility of the oligonucleotide, either through a gel as in sequencing, or through various compartments of a cell. Also, the means of attachment, typically through a 6-carbon linker, positions the probe at some distance from the other bases and allows movement of the probe in ways unrelated to the movement of the oligonucleotide. This can distort fluorescent depolarization measurements.
Ideally, a fluorescent nucleoside analog should closely resemble the naturally occurring purine or pyrimidine base structure. In particular, the probe should be attached to the oligonucleotide through the native deoxyribose chain. This keeps the probe aligned with other bases in the oligonucleotide and allows it to move in a more native-like manner. The analog should especially possess similar hydrogen bonding interactions. One type of nucleoside analog is a furanosyl pteridine derivative. Pteridines are a class of bicyclic planar compounds, some of which are highly fluorescent and are structurally similar to purines (see, FIG. 1). In fact, the fluorescence of many pteridine nucleoside analogs is known. U.S. Pat. No. 5,525,711, herein incorporated by reference, discloses pteridine nucleotide analogs as fluorescent DNA probes.
The present invention provides fluorescent nucleoside analogs that closely resemble the naturally occurring purine base structure These pteridine nucleotides are much more stable and possess higher quantum yields than prior art compounds. As such, the present invention provides pteridine nucleotides of Formula I: 
In Formula I, R1 is a functional group including, but not limited to, hydrogen and optionally substituted C1-C6-alkyl.
In Formula I, R2 is a functional group including, but not limited to, amino and mono- or di-substituted amino wherein the substituent(s) is a protecting group.
In Formula I, R3 is a functional group including, but not limited to, optionally substituted C1-C6-alkyl.
In Formula I, R4 is a functional group including, but not limited to, hydrogen and a compound having formula L. 
In Formula L, R5 is a functional group including, but not limited to, hydrogen, hydroxyl and substituted hydroxyl wherein the substituent is a protecting group.
In Formula L, R6 is a functional group including, but not limited to, hydrogen, phosphoramidite, an H-phosphonate, a methyl phosphonate, a phosphorothioate, a phosphotriester, a hemisuccinate, a hemisuccinate covalently bound to a solid support, a dicyclohexylcarbodiimide, and a dicyclohexylcarbodiimide covalently bound to a solid support.
In Formula L, R7 is a functional group including, but not limited to, hydrogen, a phosphate, a triphosphate, and a protecting group. In addition, R1 and R4 are not simultaneously L.
Compounds of Formula I are highly fluorescent under normal physiological conditions, and suitable for use in the chemical synthesis of oligonucleotides.
In another embodiment, the present invention relates to oligonucleotides that incorporate these pteridine nucleotides.
In yet another embodiment, the present invention relates to pteridine nucleotide triphosphates that may be utilized in various nucleic acid amplification processes. When used in a nucleic acid amplification process, the pteridine nucleotide triphosphates are directly incorporated into the amplified sequence rendering it fluorescent.
In still yet another embodiment, this invention relates to methods of detecting the presence, absence, or quantity of a target nucleic acid. The methods involve probing the target nucleic acid with a nucleic acid probe identical in sequence to the target sequence with the addition of the pteridine probe that does not have a pairing partner. When annealing occurs, the pteridine probe is squeezed out of the base stacking into a loop. This removes the pteridine probe from the quenching effects of base stacking and yields an increase in fluorescence intensity.
In one preferred embodiment, the loop in the above probe ranges in length from about 1 to about 100 nucleolides when the probe hybridizes to the target nucleic acid. In particularly preferred probes, the loop is an insertion in the nucleic acid probe that is otherwise complementary to the target nucleic acid or to a contiguous subsequence of the target nucleic acid. In some preferred embodiments, the insertion is three nucleotides in length and which two nucleotides are each adjacent to the fluorescent nucleotide. In particularly preferred embodiments, at least one nucleotide adjacent to the fluorescent nucleotide is a purine (e.g., adenosine), and in still more preferred embodiments, the fluorescent nucleotide is bordered by at least two adjacent purines (e.g., adenosine) in both the 5xe2x80x2 and 3xe2x80x2 direction. In a most preferred embodiment, the insertion is a single base insertion; a pteridine nucleotide of Formula I.
In yet another embodiment, the insertion is self-complementary and forms a hairpin in which the fluorescent pteridine nucleotide is present in the loop of the hairpin and does not participate in complementary base pairing. The nucleotides comprising the loop can be selected such that they are not complementary to the corresponding nucleotides of the target nucleic acid when the probe is hybridized to the target nucleic acid and where the probe is complementary to at least two non-contiguous subsequences of the target nucleic acid.
In still yet another embodiment, the invention also provides kits for performing nucleic acid amplifications or for detecting the presence absence or quantity of a nucleic acid in a sample. The kits comprise a container containing any of the probes or label oligonucleotide having a compound of Formula I described herein. The kit can further comprise, a buffer, and/or any of the other reagents useful for practicing the method to which the kit is directed. These and other embodiments of the present invention will be described in detail below.